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Published on: August 8, 2022
Comparison of the effects of a truncating and a missense MYBPC3 mutation on contractile parameters of engineered
Paul J M Wijnker1, Felix W Friedrich2, Alexander Dutsch2
1Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany; Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands.
Insights
Engineered heart tissues reveal that MYBPC3 gene mutations cause hypertrophic cardiomyopathy (HCM) by impairing cardiac myosin-binding protein-C (cMyBP-C) function, with distinct mechanisms for missense and truncating variants. Haploinsufficiency impacts function if cMyBP-C levels drop below 73%.
Area of Science:
- Cardiovascular Genetics
- Molecular Cardiology
- Biomedical Engineering
Background:
- Hypertrophic cardiomyopathy (HCM) is a genetic heart disease often caused by mutations in the MYBPC3 gene, which encodes cardiac myosin-binding protein-C (cMyBP-C).
- Understanding the precise pathomechanisms of different MYBPC3 mutations is crucial for developing targeted therapies.
Purpose of the Study:
- To compare the pathomechanisms of a MYBPC3 truncating mutation and a missense mutation using engineered heart tissues (EHTs).
- To investigate the direct effects of these mutations on cardiac contractility and protein localization within the sarcomere.
Main Methods:
- Engineered heart tissues (EHTs) were generated from Mybpc3-knockout (KO) and wild-type (WT) mouse cardiac cells.
- WT and mutant MYBPC3 were expressed in KO EHTs using adeno-associated viruses at varying multiplicities of infection (MOI).
- Contractile force, calcium sensitivity, and cMyBP-C localization were analyzed.
Main Results:
- KO EHTs exhibited increased maximal force and calcium sensitivity compared to WT.
- Expression of WT-MYBPC3 at MOI-300 (≥95% cMyBP-C level) prevented the KO phenotype, unlike MOI-100 (~73% cMyBP-C).
- Both MYBPC3 mutations, individually or combined with WT, failed to restore contractile force to WT levels, indicating impaired function. Truncating mutants showed disrupted sarcomere incorporation, while missense mutants integrated but still caused dysfunction, suggesting distinct pathogenic mechanisms like sarcomere poisoning.
Conclusions:
- Haploinsufficiency of cMyBP-C affects EHT contractile function when WT protein levels are below 73%.
- MYBPC3 missense and truncating mutations lead to distinct pathogenic mechanisms and do not fully restore normal cardiac function.
- EHTs provide a valuable platform for directly evaluating the impact of novel MYBPC3 gene variants in HCM.
Abstract:
Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. The most frequently mutated gene is MYBPC3, encoding cardiac myosin-binding protein-C (cMyBP-C). We compared the pathomechanisms of a truncating mutation (c.2373_2374insG) and a missense mutation (c.1591G>C) in MYBPC3 in engineered heart tissue (EHT). EHTs enable to study the direct effects of mutants without interference of secondary disease-related changes. EHTs were generated from Mybpc3-targeted knock-out (KO) and wild-type (WT) mouse cardiac cells. MYBPC3 WT and mutants were expressed in KO EHTs via adeno-associated virus. KO EHTs displayed higher maximal force and sensitivity to external [Ca(2+)] than WT EHTs. Expression of WT-Mybpc3 at MOI-100 resulted in ~73% cMyBP-C level but did not prevent the KO phenotype, whereas MOI-300 resulted in ≥95% cMyBP-C level and prevented the KO phenotype. Expression of the truncating or missense mutation (MOI-300) or their combination with WT (MOI-150 each), mimicking the homozygous or heterozygous disease state, respectively, failed to restore force to WT level. Immunofluorescence analysis revealed correct incorporation of WT and missense, but not of truncated cMyBP-C in the sarcomere. In conclusion, this study provides evidence in KO EHTs that i) haploinsufficiency affects EHT contractile function if WT cMyBP-C protein levels are ≤73%, ii) missense or truncating mutations, but not WT do not fully restore the disease phenotype and have different pathogenic mechanisms, e.g. sarcomere poisoning for the missense mutation, iii) the direct impact of (newly identified) MYBPC3 gene variants can be evaluated.
